Long-chain polyunsaturated fatty acids, including those of the omega-3 family which are also known as ω-3 (“omega-3”) PUFAs are interesting fatty acids in nature. They are important constituents of phospholipids that play a role in decreasing membrane rigidity. Eicosapentaenoic acid (EPA) is a major constituent of the human brain's phospholipids and serves as precursor of prostaglandins and resolvins. Another important PUFA of the omega-3 family is docosa hexaenoic acid (DHA). Improved cognitive and behavioural function in infant development seems correlated to high levels of this compound. For omega-3 PUFAs, and in particular for DHA and EPA, beneficial health effects have been shown e.g. the prevention of cancer, rheumatoid arthritis, cardiovascular diseases, the improvement of immune function, and eye and brain health (Teale, M C (ed.) 2006, “Omega-3 fatty acid research”, Nova Science Publishers. New York, and references therein). Because of these beneficial properties omega-3 PUFAs are being used extensively as nutritional lipids in health and dietary supplements and as functional ingredients in a wide range of foods. Omega-3 PUFAs presently comprise one of the biggest and strongest growing market segments in the food and beverage industry sector, with substantially increasing demand over the past years. These days, fish oil is the most abundant and widely used natural source for omega-3 fatty acids, but named source suffers from over fishing, lack in high grade oil supply with sufficient content of DHA/EPA, and quality issues (smell, formulation challenges etc.). Alternative processes involving algae and oomycetes as producer organisms are established or under development (Hinzpeter, Grasas y Aceites (2006) 57:336-342; Ward, Process Biochemistry (2005) 40:3627-3652). Since the supply of fish oil of high quality is increasingly limited, it was attempted to find alternative, sustainable biological sources.
Various groups of marine algae have been explored for over 20 years and some products based on algal biomass have meanwhile entered the market. Some oomycetes belonging to the group of stramenopiles (a group of algae-like eukaryotic organisms also known as “Chromophyta”) were also occasionally reported to produce the above mentioned compounds (e.g. of the genera Achyla and Pythium; (Aki, J. Ferm. Bioeng. (1998) 86:504-507; Cheng, Biores. Technol. (1999) 67:101-110; Athalye, J. Agric. Food Chem. (2009) 57:2739-2744). In other stramenopiles (e.g. the genera Schizochytrium and Thraustochytrium; as described in U.S. Pat. No. 7,022,512 and WO2007/068997) and in species of the dinoflagellate Amphidinium (US 2006/0099694), DHA may represent up to 48% of the fatty acid content of the cells, which are the highest contents so far known in the Eukaryota. However, the cultivation of these organisms in industrial scale still poses a challenge even after several years of development. Other alternative biological sources for omega-3 PUFAs hitherto found are prokaryotic eubacteria [Nichols, Curr. Opin. Biotechnol. (1999) 10:240-246; Gentile, J. Appl. Microbiol. (2003) 95:1124-1133]. However, the commercial exploitation of these organisms for PUFA production on an industrial scale is hampered by the slow growth characteristics of these psychrophilic microorganisms, as well as their inherently low yields and productivity. Heterologous expression of Omega-3 PUFA gene clusters in suitable, industrial organisms constitutes a valid alternative to the production of the desired Omega-3-PUFAs at an industrial scale, which has manifold advantages to production processes using the wild type strains. It has been established for a long time that Omega-3-PUFAS are biosynthesized in a similar manner as the polyketide secondary metabolites in both prokaryotic and eukaryotic organisms (see overview by Metz, Science (2001) 293:290-293), which allows for utilization of similar methods techniques as those that have been established in microbial biotechnology in order to improve and modify production of antibiotics and anticancer agents. For myxobacteria, such work regarding evaluation of secondary metabolites biosyntheses has been described and outlined in recent reviews. (Wenzel, Curr. Opin. Biotechnol. (2005), 16: 594-606; Wenzel, Curr. Opin. Drug Discov. & Develop. (2009), 12 (2): 220-230; Wenzel, Nat. Prod. Rep. (2009), 26 (11): 1385-1407).
The organization of the PUFA genes in gene clusters allows for their cloning and transfer into heterologous hosts, which can per se be either prokaryotic or eukaryotic organisms.
Gene clusters encoding synthetic pathway enzymes for biosynthesis of omega-3 PUFAs are known from various marine bacteria, including species of the genera Moritella (Tanaka, Biotechnol. Lett. (1999) 21:641-646; Morita, Biochem Soc Trans 28:943-945 (2000)), Photobacterium (Allen, Microbiology (2002) 148:1903-1913), and Shewanella (Lee, J. Microbiol. Biotechnol. (2009) 19:881-887). Such bacterial omega-3 PUFA biosynthetic gene clusters were already transferred to and expressed in Escherichia coli (Lee, Biotechnol. Bioproc. Engin. (2006) 11:510-515; Orikasa, Biotechnol. Lett. (2006) 28:1841-1847; Orikasa, Biotechnol. Lett. (2007) 29:803-812). Furthermore, heterologous EPA production was enhanced by substitution of promoter sequences within the biosynthesis gene cluster (Lee, Biotechnol. Lett. (2008) 30:2139-2142). The EPA gene cluster from a Shewanella sp. was also expressed in the transgenic marine cyanobacterium of the genus Synechococcus (Takeyama, Microbiology (1997) 143 (Pt 8):2725-2731. Orikasa, Biotechnol. Lett. (2007) 29, 803-809) observed enhanced heterologous production of EPA in E. coli cells that co-express EPA biosynthesis genes and foreign DNA fragments including a high-performance catalase gene. The yields of EPA were augmented from 3% to 12% of the total fatty acid content. Jiang (Methods in Enzymology (2009) 459, 80-96) have summarized previous research on the characterization of the important tandem acyl carrier protein domains in polyunsaturated fatty acid synthases, also accounting for several other studies that involved heterologous expression of the respective genes in E. coli. These examples show that it is feasible to attain heterologous production of bacterial Omega-3 PUFA genes, even though no commercial product based on such techniques has so far resulted. Myxobacterial Omega-3 PUFA gene clusters were so far never identified and thus not expressed heterologously. Myxobacterial natural product biosynthetic pathways have been successfully expressed in a variety of heterologous hosts including Pseudomonas putida (Gross, Chem. Biol., (2006) 13: 1253-1264; Wenzel, Chem. Biol. (2005) 12 (3): 349-356).
Due to the great commercial significance of these products, there are various examples for the feasibility of heterologous expression of genes involved in biosynthesis of omega-3 PUFAs from various eukaryotic and prokaryotic organisms in transgenic plants and fungi. As exemplified by Domergue (Plant Physiol. (2003) 131, 1648-1660) and the fatty acid biosynthetic genes of the stramenopile alga, Phaeodactylum tricornutum, even the elucidation of the biosynthesis of certain marine biological sources that are not easy to cultivate in the laboratory have often involved their heterologous expression in Saccharomyces cerevisiae or other yeasts. Cipak (Free Radic. Biol. & Med. (2006) 40, 897-906) expressed a desaturase gene from the rubber tree Hevea brasiliensis also in S. cerevisiae. Tonon (2005, Plant Physiol. 138, 402-408) used the same host to express and characterize a acyl-coenzyme A (acyl-CoA) synthetase that was found from genome mining in the diatom, Thalassiosira pseudonana. Hsiao (2007, Mar. Biotechnol. 9, 154-165) also used S. cerevisiae as host for heterologous expression and functional characterization of a delta-6 desaturase from the marine microalga Glossomastix chrysoplasta. Lee (2008, Biotechnol. Bioproc. Engin., 13, 524-532) have demonstrated successfully the activity of delta-9 elongase, a crucial enzyme in Omega-3 PUFA biosynthesis from the stramenopile, Thraustochytrium aureum by heterologous expression in the yeast Pichia pastoris. Li (Biotechnol. Lett., 31, 1011-1017) reported an improvement of arachidonic acid and eicosapentaenoic acid production by increasing the copy number of the genes encoding fatty acid desaturase and elongase from the alga Phaeodactylum tricornutum in Pichia pastoris as heterologous host; however, only relatively low percentages of the PUFAs of 0.1 and 0.1%, respectively of the total fatty acid content of Pichia pastoris were attained. The latter yeast, as well as Yarrowia lipolytica, Hansenula anomala, and other methylotrophic and/or oleaginous yeasts, appear ideal for production of PUFAs as they are well-established industrial producer organisms (Banlar, Appl. Microbiol. Biotechnol. (2009) 84, 47-865; Silva, J. Food, Agric. & Environment. 2009, 7, 268.273). Moreover, oleaginous yeasts, which are especially preferred production organisms and which are capable of growing in highly lipophilic environments may accumulate very large amounts of lipids, and can use a broad range of hydrocarbons as substrates. Other applications of yeast-like fungi in industrial microbiology and biotechnology have been summarized by Porro (2005, Mol. Biotechnol. 31, 245-259) and Idiris (2010, Appl. Microbiol. Biotechnol. 86:403-417).
Graham (Curr. Opin. Biotechnol. (2007) 18 (2), 142-147) have summarized the state of the art in metabolic engineering of transgenic plants for biosynthesis of omega-3 PUFAs. They emphasized in particular the recent progress in the rational design of oilseed crops with high content of Omega-3 PUFAS but also discussed several factors that may be responsible for the hitherto observed, inherent low yields, which so far prevented the development of competitive production processes for PUFAs by using oilseed or other seed plants. Taylor (Plant Biotechnol. J. (2009) 7, 925-938) have recently identified and cloned 3-ketoacyl-CoA synthase from Cardamine graeca and reported its heterologous expression in Brassica oilseeds, resulting in increased production rates of the PUFA, nervonic acid and reported a 15 fold increase of nervonic acid in the heterologous host. However, nervonic acid is an omega-9, rather than an omega-3 PUFA and has by far not reached the commercial importance of DHA and EPA. In addition, nervonic acid is derived from elongation of oleic acid in nature.
Lee et al (Biotech. Bioproc. Eng. (2006) 11, 510-515) describe the heterologous expression of a putative polyketide synthetase gene cluster from Shewanella oneidensis MR-1 for production of EPA in E. coli. The gene cluster is identified only as a 20.195 kb DNA fragment obtained by long PCR using a primer pair, the nucleotide sequences of which are given, and by the sizes of restriction fragment generated by BglII and NdeI.
Orikasa et al (Biotechnol. Lett. (2006) 28, 1841-1847) describe synthesis of DHA in E. coli by expression of a gene cluster from Moritella marina MP-1.
Schneiker et al (nature biotechnology (2007) 25, 1281-1289) describe sequencing the complete genome of Sorangium cellulosum and identify polyketide synthetase gene clusters involved in synthesis of single antibiotic compounds. A comparison to the Myxococcus xanthus genome revealed a lack of global synteny.
Dickschat et al (Org. Biomol. Chem. (2005) 3, 2824-2831) describes the analysis of the fatty acid profile of Stigmatella aurantiaca using GC-MS, and the synthetic steps of biosynthesis.
There is a demand for methods for the production of PUFAs, particularly PUFAs containing 3, 4 or more double bonds, which methods biosynthesize the PUFAs de novo, rather than modify, e.g. elongate, other fatty acids which in turn serve as precursors.